ABSTRACT Native top down mass spectrometry (nTDMS) is a multi-tiered tandem mass spectrometry technique able to characterize proteoforms and intact protein complexes in their native state and represents a potentially revolutionary technology still in the ?early adopters? stage of the technology life cycle. By utilizing gentle sample preparation and analysis methods, nTDMS can provide a direct measure of the endogenous noncovalent protein-protein and protein-ligand interactions critical for biological regulation. In addition, with the new wave of therapeutic protein molecules, nTDMS is being relied upon more and more to provide answers to different biotechnology questions. Recent instrument advances have widely increased the accessibility to nTDMS by enabling more mass spectrometers to be capable of nTDMS experiments. Overall, these applications and technology improvements have led to a rapid rise in the number of researchers using nTDMS. However, the advances propelling nTDMS have not been matched with software advances to handle these new datasets, which contain many non-covalent ligand and cofactor binding events as well as information on the subunit stoichiometry of protein complexes. As such, users have had to manually process much of the data, ultimately holding back nTDMS from reaching its true potential. Proteinaceous has previously developed data analysis solutions to uncover important biological information from denaturing mass spectrometry analyses of intact proteins and now proposes a software named ProSight Native to address the pressing bioinformatics needs of nTDMS scientists. ProSight Native will get in at the ground-floor of nTDMS as the first software directly developed to serve as a complete nTDMS analysis package, from determination of protein complex subunit and cofactor stoichiometry down to full proteoform characterization at the subunit level. In this SBIR, Proteinaceous will implement two critical features for bioinformatics analysis of data produced by nTDMS: handling of ?native? modifications and noncovalent ligands and the computational characterization and scoring of protein complex stoichiometry. These new features will be thoroughly validated using data from the largest repository of nTDMS protein complexes published to date, providing a robust software platform that can process the complexity of data generated from both academia and industry. The work proposed in Phase I provides a strong initial foundation for further advancements during Phase II, including more advanced scoring methodologies accompanied with high-throughput searching. In summary, ProSight Native will serve as an analysis tool focused on characterizing intact protein complexes from data produced by nTDMS and will enable new and valuable insights in biological and biotechnology research.